Method for determining the coagulation potential of a plasma sample

ABSTRACT

A method of determining the coagulation potential of a plasma sample be pre-incubating the plasma sample with a reagent such that endogenous protein C in the plasma is at least partially converted into activated protein C by the reagent, adding factor Xa which is progressively inactivated by antithrombin III/heparin cofactor 2 during the preincubation, adding an exogenous reagent which activates factor X to Xa or prothrombin to thrombin in a factor V-dependent manner, monitoring a reaction indicative of the rate of coagulation of the plasma sample, comparing that rate of coagulation control, or the equivalent rate determined for an individual without impaired coagulation control, or the equivalent rate determined for the plasma sample in the absence of protein C activator, and determining the coagulation potential of the plasma sample from one or other of the compairsons.

This application is a U.S. nation stage application filed under 35U.S.C. §371 based on international application No. PCT/AU99/00069 filedFeb. 1, 1999, which claims priority from Australian application No. PP1596 filed Feb. 2, 1998.

TECHNICAL FIELD

The present invention relates to an improved test for measuring bloodcoagulation potential of patients' plasma for the purpose of predictingrisk of thrombosis.

BACKGROUND ART

Mechanisms for blood coagulation, thrombosis and haemostasis are welldescribed in International Patent Publication WO 91/01382 the contentsof which are incorporated herein by reference.

It is known from International Patent Publication WO 93/01261 andpublications by Bertina et al 1994 and Dahlback et al 1995 that the riskof thrombosis in patients with a mutant factor V molecule known as theLeiden variant, or with activated protein C impairment for some otherreason, may be determined by activating the coagulation system in aplasma sample and incubating the sample with activated protein C in whathas come to be known as an activated protein C impairment, impedance orresistance test. There are precedents for this test in which impairmentof activated protein C has been detected in patents with acquiredthrombophilia (Mitchell et al, 1986; Amer et al. 1988).

New tests have recently been proposed to screen for most defects in theprotein C pathway (PCP) thereby to rationalise the approach toindividual assays for protein C, S and factor V(Leiden) which arecurrently requested together in all cases of thrombophiliainvestigation, with a very low rate of abnormality finding. These2-stage clotting tests usually involve activating the patient's ownplasma protein C either with thrombin/thrombomodulin complex or theactivator from Agkistrodon Contortrix venom, commonly referred to asPROTAC™ of Pentapharm, Basle. This activated protein C (APC) theninactivates the patient's own factor Va in a protein S-dependent mannerduring a subsequent clotting test, yielding longer clotting times thanif protein C had not been activated. Clotting times shorter than normalare obtained when defects in protein C and protein S occur as well aswhen APC resistant factor V(Leiden) is present. Such tests have beendescribed based oil Activated Partial Thromboplastin Times (APTT) eg AU28416/95, EP 718628 “Method for diagnosis of blood coagulationdisorders”, dilute prothrombin time tests (PT) and WO 96/42018“Thrombosis risk test”.

A substrate conversion reaction rate may be determined by thecoagulation time or by the time required for the conversion of achromogenic substrate to a coloured product. The conversion rateobtained is compared with values obtained in the absence of protein Cactivator or PCA and also with results on normal plasma samples. If thecoagulation time is not sufficiently prolonged by protein C activator,it indicates that the individual from which the sample is derived may beat a higher-than-normal risk of thrombosis.

It is well known that activation of endogenous protein C in plasma bythe activator from A. Contortrix venom prolongs subsequent clottingtimes to a degree related to the protein C content. Several otherfactors, however, influence or interfere with this test. These factorsinclude protein S, factor V(Leiden) which are now recognised asthrombotic risk factors in their own right.

The present inventor has recently developed an improved APC resistancetest which is described in WO 96/04560. This test requires the additionof exogenous reagents which activate factor V and activate the commonpathway of the blood coagulation mechanism through factor X or byinducing the formation of thrombin in a factor V dependent mannertogether with exogenous APC to a plasma sample. It was found that iffactor V is specifically activated by an exogenous reagent in additionto activation of the common pathway through factor X, the test for APCresistance may be made more sensitive and specific than previously knowntests. The present inventor has also found that improved specificity isobtained when a complex factor X activator is used together with thefactor V activator. This test, because the Russells viper venom containsactivators of both factor X and factor V, has been referred to as theRussells Viper Venom (RVV)-based test. A similar result is achieved ifprothrombin is activated to thrombin by a factor V dependent activatorin the presence of a factor V activator such as those from Australianelapid venoms.

The protein C pathway is one of a number of antithrombotic mechanismsoperating within normal blood vessels to control coagulation and preventthrombosis. Probably the most important of these mechanisms is theglycosaminoglycan (GAG) pathway which requires antithrombin III as acofactor and heparin cofactor 2. Thrombin and factor Xa are controlledby these two plasma inhibitors which are modulated by glycosaminoglycanssuch as heparin sulphates normally on endothelial cells lining healthyblood vessels.

The present inventor has made the surprising finding that such tests maybe further modified to allow improved discrimination between healthyindividuals and patients with impaired or aberrant blood anti-thromboticmechanisms.

DISCLOSURE OF INVENTION

In a first aspect, the present invention consists in a method ofdetermining the coagulation potential of a plasma sample, the methodcomprising the steps of:

-   -   (a) preincubating the plasma sample with a reagent such that        -   (i) endogenous protein C in the plasma is at least partially            converted into activated protein C by the reagent, and        -   (ii) adding factor Xa which is progressively inactivated by            antithrombin III/heparin cofactor 2 during the            preincubation;    -   (b) adding to the preincubated plasma sample (a) reagents to        initiate clotting comprising:        -   (i) an exogenous reagent which activates factor X to Xa or            prothrombin to thrombin in a factor V-dependent manner, and    -   (ii) components, such as phospholipid and calcium ions, that are        necessary for efficient coagulation of the plasma sample;    -   (c) monitoring a reaction indicative of the rate of coagulation        of the plasma sample;    -   (d) comparing the rate of coagulation detected in step (c) with        the equivalent rate determined for a normal patient, or        comparing the rate of coagulation detected in step (c) with the        equivalent rate determined for the plasma sample in the absence        of protein C activator; and    -   (e) determining the coagulation potential of the plasma sample        from one or other of the comparisons of step (d).

The reagent used in step (a) preferably also contains low levels ofglycosaminoglycans such as regular or low molecular weight heparins,dermatan or dextran sulphates in addition to factor Xa The inclusion ofthese components to the reagent makes the test more sensitive toantithrombin III.

Preferably, the reagent used in step (a), which transforms protein Cinto activated protein C, is diluted substantially whole snake venom,preferably diluted snake venom from Agkistrodon Contortrix, or relatedspecies such as A. Piscovorus, A. Bilineatus, A. C. Laticinctus, A. C.Moccason. It has been found that by selecting an appropriateconcentration of the snake venom, it is possible to obtain a diagnosisof impaired anticoagulation by the one test. A protein C pathway (PCP)ratio of below a pre-determined value can be indicative of impairedcoagulation control in the patient's plasma. When using A. Contortrixwhole venom diluted at a concentration of about 0.002%, it is possibleto differentiate between plasma from normals, whether these come fromhealthy or pregnant or lupus anticoagulant positive individuals andplasma from individuals with thrombotic risk factors such as APCresistant factor V(Leiden) and protein C deficiency. A PCP ratio in thisinstance of below about 2 would be positive in the present test.Similarly for a concentration of 0.003%, a value of below 2.5 would bepositive (see FIG. 1).

Preferably, the incubation in step (a) is carried out at neutral orslightly basic conditions, more preferably at about pH 7.5. Theincubation is carried out for sufficient time for activation of theprotein C in the plasma. Typically incubation times of around 5 minutesas usual for the preincubation interval in most automated APTT testmethods have been found to be sufficient.

The present inventor has made the surprising finding that the protein Cactivator purified from A. Contortrix venom (a commercial product“Protac™” available from Pentapharm AB (Switzerland)) does not work verywell in the present invention (see FIG. 2). The present inventor hasfound that dilute A. Contortrix venom is particularly suitable. It ispossible that the purification process used to produce this commercialprotein C activator removes an additional activator or agent that ispresent in whole venom which is preferably required for the presentinvention. The precise nature of the ingredient is not yet clear,however, it would appear to be a procoagulant unaffected by deficiencyof vitamin K-dependent factors or Warfarin treatment. It will beappreciated that this additional activator or agent could also bepurified from whole venom and combined with any commercially availablepurified protein C activator for use in the present invention. Theindividual active fractions may also be purified and recombined toproduce a reagent suitable for the present invention.

Factor Xa of either human or animal origin can be included and incubatedwith the protein C activator reagent. This factor can be formed fromendogenous factor X by venom activators Factor Xa tends to shortenclotting time. Thus the level of Russells viper venom which needs to bepresent ill the second reagent (with the calcium and phospholipid) toyield clotting times of 80-120 seconds on normal plasma, similar tothose in regular protein C pathway tests, can be proportionally reduced.Also, heparin or glycosaminoglycans can be included in the preincubationreagent to enhance the interaction between antithrombin III and factorXa to enhance sensitivity to low levels of antithrombin III.

In a preferred form of the present invention, the patient's plasmasample is incubated with an exogenous activator for protein C and factorX. The exogenous activator of protein C is preferably highly diluted andunfractionated Agkistrodon contortrix venom. The factor X activator ispreferably derived from the venom of Russell viper (Vipera Russelli) andother immunologically cross-reactive species. The snake venoms mayeither be used in a diluted but unfractionated form which contributes tothe simplicity of the test or, preferably, may be used in a fractionatedform utilising isolated venom components.

Rather than directly activating factor X with an exogenous reagent inthe second stage of such tests one may also obtain an improvement overthe known activated protein C test by utilising an exogenous reagentthat induces in the plasma the presence of thrombin in a factor Vdependent manner. In this aspect of the invention factor V dependentprothrombin activators such as those from certain Australian Notechisand Pseudonaja venoms, such as Pseudonaja Textilis, Notechis Scutatusand Oxyuranus Scutellatus, may be used. The use of this system by-passesfactor X and all factors above it thereby making the test more specificthan that based on Russells viper venom alone. The use of additionalvenom-derived factor V activators is desirable exactly as describedabove for the Russell viper venom activated system which involves factorX activation.

In one embodiment of the invention, the components in step (b) withwhich the patient's plasma and its pre-incubants are to be mixed arecombined into a single mixture. Such a single mixture preferably alsocontains supplemental components such as suitable buffers andpreservatives. In addition the mixture preferably contains polybrene oranother similar agent to reverse the effect of any heparin that may bepresent in the test samples or which may be added in the preincubationreagent (i). The incubation mixture preferably also contains relativelyhigh levels of phospholipid at high ionic strength to overcomenon-specific inhibitors such as lupus anticoagulants that may be presentin the plasma sample.

Another complicating feature in test plasma samples may be the defectcaused by oral anticoagulants. Many such thrombotic patients may alreadybe on oral anticoagulant treatment and this affects the coagulationtests currently used to assess activated protein C resistance. Theconventional method for minimising such interference is by mixing testplasma with factor V deficient plasma. The present invention, however,does not necessarily require such manipulation as such antithromboticagents if used within their therapeutic range do not necessarilyadversely effect the test. The rationale behind this is apparent fromFIG. 1.

In another embodiment of the present invention, factor Xa may be used inthe preincubation reagent at such a high level that no additionalRussells viper venom may be required in the second mixed reagent(comprising then only phospholipid and calcium) to yield an idealclotting time of 100 seconds with normal plasma (intended range of50-200 seconds). In this case, the clotting time should be mainlyaffected by levels of autithrombin 111 (ATIII) and heparin cofactor 2(HCF2) and not by protein C or S nor by the presence of factorV(Leiden). In this scenario, the method could be referred to as a testfor the glycosaminoglycan pathway or a “GAG” test, The GAG test servesas a complimentary role to PCP tests as a preliminary screening test forlikely defects in ATIII and HCF2, though in practice, it appears poorlysensitive to HCF2 (see FIG. 4). This may not be a problem, however, asHCF2 in fact is of doubtful importance as a thrombotic risk factor incomparison to ATIII.

Most tests for ATIII and HCF2 presently used require a preliminary highdilution of the test plasma to be carried out. This is usually in abuffer containing high levels of heparin or GAGs to facilitate completeinteraction of thrombin or factor Xa with ATIII or HCF2. Thequantitative loss in thrombin or factor Xa enzyme activities is thenconverted to functional ATIII or HCF2 present in the test plasma.However, if a single thrombophilia screening test proves to be sensitiveenough to all the known thrombotic risk factors in diagnostic practice,then an appropriate mixture of ACCV/PCA and factor Xa in thepreincubation reagent and dilute Russells vipervenom/phospholipid/recalcifying reagent to provide equal sensitivity toall the known thrombotic risk factors would be preferred.

The detection system for monitoring the potential rates of change withinthe coagulation system may be a coagulation time assay or a chromometricor fluorometric assay using an appropriate synthetic substrate. Suchdetection systems are well known and described in the patentspecifications referred to in the introductory portions of thisspecification.

Some patients' plasma may give borderline results when assayed by themethod according to the present invention such that it is not possibleto determine unequivocally between “normal” and factor V(Leiden)deficient plasma samples. The present inventor has made the surprisingdiscovery that diluting these “borderline” samples with low ionicstrength solutions including water and carrying out the method accordingto the first aspect of the present invention can differentiate betweennormal and factor V(Leiden) samples. The method according to the firstaspect of the present invention also provides improved discrimination ofFVL heterozygotes from homozygous individuals.

In a second aspect, the present invention consists in method todifferentiate between patients with factor V(Leiden) from normalindividuals, the method comprising diluting plasma from the patients andthe normal individuals with low ionic strength solutions including waterand repeating the method according to the first aspect of the presentinvention.

Preferably, the plasma are diluted 1:1 with water, preferably distilledor filtered water, prior to repeating the coagulation assay. The factorV(Leiden) plasma will usually have ratios equal to or less than theratios obtained when undiluted. Furthermore, the ratios obtained for thefactor V(Leiden) plasma will usually be less than the ratios obtainedfor normal plasma assayed with the same test conditions. Prior to thepresent invention, it would have been necessary to add factor Vdeficient plasma to all plasma test samples and then re-assay forclotting abnormalities.

The use of low ionic strength solutions, and particularly distilledwater, is significantly cheaper than factor V deficient plasma that isrequired in tests currently used. Furthermore, low ionic strengthsolutions, and particularly distilled water, are far easier to sourcethan factor V deficient plasma. The present invention therefore offers areal advantage in cost and tests requiring factor V deficient plasmapresently in

In a third aspect, the present invention consists in a method of testingantithrombin III deficiency in a plasma sample, the method comprisingthe steps of:

-   -   (a) preincubating a first sample of a test plasma with factor        Xa:    -   (b) adding to the preincubated first test plasma a reagent to        initiate clotting and measuring the clotting time of the        preincubated test plasma;    -   (c) adding to a second sample of the test plasma a reagent to        initiate clotting and measuring the clotting time of the second        test sample; and    -   (d) comparing the clotting times of the first and second test        samples, wherein a shorter clotting time in the first test        sample being indicative of antithrombin III deficiency in the        plasma sample.

Preferably, in step (a) the first test plasma is preincubated with anequal volume of factor Xa in buffer for around 5 minutes at 37 degC.More preferably, ill step (a) test plasma (0.1 ml) is preincubated withan equal volume of factor Xa (optimally 0.002 u/ml or higher if GAGs areadded) in 0.02 M HEPES buffer at pH 7.2 for 5 minutes at 37 degC.

Preferably in step (b) a further equal volume of calcium chloridecontaining soybean lecithin is added to initiate clotting of thepreincubated sample and the time to clot is determined. More preferablyin step (b) a further equal volume (0.1 ml) of 0.02 M calcium chloridecontaining 0.1% soybean lecithin is added to initiate clotting of thepreincubated sample. A target range of about 80-120 sec has been foundfor normals.

The above result is compared with that obtained when the same testplasma is clotted with of a mixture of the two reagents (ie. the FXareagent and the calcium chloride/phospholipid reagent). Preferably thesame test plasma (0.1 ml) is clotted with 0.2 ml of a 1:1 mixture of the2 reagents (ie. the FXa reagent and the calcium chloride/phospholipidreagent). A target range of about 30-35 sec has been found for normals.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

In order that the nature of the present invention may be more clearlyunderstood, a preferred form will be described with reference to thefollowing example and the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows effect of varying ACCV level in PCP/FVL tests on varioustest plasmas. Test plasmas preincubated for 5 minutes with varyinglevels of ACCV and then clotted with RVV/phospholipid/calcium reagent(LA-Confirm) in ACL300 in APFT mode. Showing RVV clotting time ratios(PCP ratios) plotted against the concentration of ACCV (%).

FIG. 2 shows Protac™ or whole dilute Agkistrodon Contortrix C. venom(ACCV) dilutions were preincubated with pooled normal plasma for 5minutes and then clotted with phospholipid-rich Russells viper venomreagent (LA-Confirm). Results show the RVV clotting times obtained on anACL300 clotting machine plotted against the concentration of Protac™(u/ml/20) or ACCV(ug/ml) used. (Note that PCP ratios are calculated asthe RVV clotting times with any given ACCV or Protac™level divided bythe clotting time with no activator present).

FIG. 3 shows effect of individual thrombotic risk factors on the PCPcarried out with dilute whole ACC venom. Showing PCP ratios (RVVclotting times with and without protein C activation) plotted againstlevel of each factor shown. From top to bottom; HCF2; ATIII; Prot.S,Prot.C, Factor V(Leiden). Mixes prepared from individual factordeficient or factor V(Leiden) positive (heterozygote) plasmas and poolednormal plasma, itself representing 100%.

FIG. 4 shows effect of individual thrombotic risk factors on a mixedGAG/PCP test system. Preincubation reagent contained dilute whole ACCVand 0.002 u/ml factor Xa. Reagent was mixed with each test plasma for 5minutes at 37 degC before being clotted with a reduced Russells vipervenom reagent/phospholipid/calcium reagent. Results show the ratios ofclotting times with and without preincubation plotted against factorlevel. In descending order on left axis; HCF2, Prot.S, ATIII, Prot.C,Factor V(Leiden).

FIG. 5 shows borderline PCP/FVL results. Results show scatterplot ofPCP/FVL ratios obtained on selected several warfarin patient with neatand water-diluted plasmas. Eight (8) neat plasmas all gave borderlineabnormal ratios(1.2-1.8), but after dilution with water, improveddiscrimination of FVL cases from normals was achieved.

MODES FOR CARRYING OUT THE INVENTION

Method

A method is described for a clotting test which is more specific fordetecting resistance to activated protein C due to the factor V(Leiden)mutation than the original system described by Dahlback. The methodinvolves 2 steps. In the first step, test plasma is incubated withdilute whole Agkistrodon Contortrix venom at 0.002-0.004% and pH 7.5 for5 minutes. In the second step, phospholipid-rich Russell viper venom isadded and the time required for a fibrin clot to form is determined.

A “control” or blank test to detect baseline coagulation abnormalitiesmay be carried out in exactly the same way, except that no AgkistrodonContortrix venom should be present in the first pre-incubation step.Chromogenic substrates could be used as an alternative to clot formationfor detecting the formation of thrombin, but these are more expensive.

Mechanism

It is known that Agkistrodon Contortrix venom contains an activator ofprotein C. The active component has been isolated and sold under thetrade mark “Protac™” by Pentapharm AB (Switzerland). Protac™ has beenpatented for use in tests for quantitating protein C and more recentlyin tests for assessing the function of the protein C pathway (PCP) asdescribed above. During the course of the first incubation (above)protein C in the test plasma is converted to an enzymatically-activeform (activated protein C or APC). This is a powerful anticoagulantwhich destroys factors Va and VIIIa, thereby interfering with theclotting mechanism and prolonging certain clotting tests. In individualswho are deficient in protein C or S, the anticoagulant effect of thevenom protein C activator is reduced relative to normal and the clottingtimes are shorter than normal. Also, if the patient plasma contains acommonly-occurring mutation in factor V called the FV(Leiden) variant,the clotting times are less prolonged by either activated protein C orthe venom protein C activator than with normal plasma. FV(Leiden) lacksa specific APC sensitive cleavage site involved in the inactivation ofnormal factor V and therefore it persists in such test systems andshortens the clotting times.

All of these defects interfere with the normal functioning of the PCPand are associated with clinical thrombosis. All three defects areusually detectable by a shorter than normal clotting test result in thepresence of protein C activator. Patients who are on oral anticoagulantshave reduced levels of vitamin K-dependent clotting factors as well asprotein C and S and cannot usually be screened for factor V(Leideni)which such a test. It has become conventional to mix such patient'splasma with factor V deficient plasma to “correct” all clotting factordefects and protein C and S levels prior to carrying out an APCresistance tests for factor V(Leideni).

The present iwventor has found that use of whole diluteAglistrodoiiCoiitortrLY venom (ACCV) is preferable to the use of isolated protein Cactivator in the RVV-based PCP test described in WO 96/04560 for thefollowing reasons. The test becomes insensitive to protein S deficiency(which is a less important thrombotic risk facto than FVL or protein C)and less affected by low protein C levels and more sensitive to factorV(Leiden). The effect of relatively high levels of ACCV on the RVVT ofnormal plasma seems to be similar to that of lower levels, unlike thatof the isolated activator which prolongs the RVVT to an increasingdegree with concentration. Higher levels of ACCV can be used to activatethe small concentrations of protein C found in patients on oralanticoagulants for more effect in the test and to overcome acquired APCresistance in patients taking oral contraceptives or who are pregnant.Thus by using higher levels of the whole ACCV it is possible to screenfor the Factor V(Leiden) defect in plasma from Warfarin patients,pregnancy plasma and other conditions which previously required mixingwith factor V deficient or other normalising factors.

Advantages

1. No need to mix test plasma with factor V deficient plasma for thedetection of factor V(Leiden) among complex patients.

2. Plateau concentration dependencies means higher levels of ACCV can beadded with a less prolonged normal clotting time.

Method

The improved test is based on the factor V(Leiden)-specific PCPscreening test which uses a phospholipid-rich RVV reagent. Thecomposition of the reagent has been modified to make it less sensitivethan usual to variations in Protein C and Protein S levels in thepresence of a protein C activator. Since this RVV reagent is alreadydesigned to be heparin and lupus anticoagulant resistant and since itsmechanism is through the common pathway, this test is more reliable thanthose based on APTTs and PTs.

Reagents

-   1. Protein C Activator (PCA)    -   Preparation        -   Reconstitute in volume of distilled water as indicated on            the vial        -   Gently invert to mix- DO NOT shake        -   Allow to stand at room temperature for 10 minutes before            use.-   2. PRVV Reagent (Phospholipid-rich Russell viper venom reagent)    -   Preparation        -   Reconstitute in volume of water as indicated on the vial        -   Gently invert to mix- DO NOT shake        -   Allow to stand at room temperature for 10 minutes before            use.            Reconstituted Stability

Product Conditions Time PCA 2-8° C. 48 hours 37° C. 12 hours PRVV 2-8°C. 48 hours 37° C. 12 hours −20° C.  1 month (freeze thaw only once)Specimen

Mix nine parts of freshly collected blood with one part 3.5% (0.12 M)trisodium citrate. Centrifuge as soon as possible after collectionat >1500 g for 15 minutes. Separate plasma and store at 2-8° C. Testwithin 4 hours of collection. Plasma may be stored frozen at −30° C. orbelow for up to six months.

Jaundiced, lipaemic and haemolysed specimens can give false clottingtime results. These results may also occur in patients with abnormalhaematocrits, as plasma to citrate concentration in these samples is notoptimal.

Test Procedure

Method

The PCP Test is not affected by Heparin levels of up to 0.5 IU/ml.

Test with PC Activator

-   1. Pre-warm a slight excess of PRVV reagent, allowing 0.1 ml per    test, to 37° C.+1° C. in a reagent reservoir.-   2. Dispense 0.1 ml of test plasma into a test tube.-   3. Add 0.1 ml of Activator to the test plasma and warm at 370° C.    for 5 minutes.-   4. Add 0.1 ml pre-warmed PRVV reagent and time from the moment of    addition of the reagent to a clotting end-point using the tilt tube    technique.

5. Repeat for duplicate test values and report the average of these asthe result.

Test without PC Activator

-   1. Pre-warm a slight excess of PRVV Reagent, allowing 0.1 ml per    test, to 37° C.+1° C. in a reagent reservoir.-   2. Dispense 0.1 ml of test plasma into a test tube.-   3. Add 0.1 ml of distilled water to the test plasma and warm at    37° C. for 5 minutes.-   3. Add 0.1 ml pre-warmed PRVV Reagent and time from the moment of    addition of the reagent to a clotting end-point using the tilt tube    technique.-   5. Repeat for duplicate test values and report the average of these    as the result.

The results of PCP tests of varying A. Contortrix whole venom levels onvarious test plasma are shown in FIG. 1. The use of levels of between0.002 to 0.004% ACCV in the test allows the differentiation between serafrom normal individuals (PNP1, PNP2 and PNP3), oral anticoagulant pool(O/A pool) and pooled sera from pregnant individuals(PREG.POOL), frompatients with impaired clotting function (FV(L) and FV(L)+O/A). PCPvalues of below 2 and 2.5, respectively for tests using 0.002 and 0.004%ACCV are seen to be indicative of impaired clotting.

FIG. 1 shows the protein C pathway (PCP) clotting time ratios (clottingtimes with protein C activator present/those without activator) obtainedon a series of patients and normals using increasing levels ofAgkistrodon Contortrix Contortrix Venom (ACCV). Normal plasmas show thelargest effect initially, but seen to dip down at ACCV levels above0.002%. Oral anticoagulant-treated, factor deficient (partially aluminaadsorbed) and pregnancy plasmas show a more gradual increase with noevidence for a dip. Factor V(Leiden) positive plasmas all remain low.Thus, by selecting an appropriate ACCV level of approximately 0.003% itis possible accomodate all the FVL negative cases within a tight PCPratio range, representing the normal or reference range, regardless ofseveral other complicating factors. (Note that this RVV-based system isalready insensitive to therapeutic levels of heparin and to anyabnormalities involving factors above factor X in the clotting pathway.)

FIG. 2 shows a direct comparison of Protac™ with Agkistrodon Contortrixvenom (ACCV) in the PCP test. Increasing levels of both protein Cactivators were added to pooled normal plasma prior to testing with thephospholipid-rich dilute RVV reagent. Both activators resulted insimilarly prolonged clotting times at levels below 10 ug/ml for ACCV or0.5 u/ml for Protac. However, above this concentration the Protac™ gavefurther prolongation, whereas the ACCV gave shorter clotting times. Thisis the reason for the unexpected “dips in the PCP ratios for normalplasma in FIG. 1 at ACCV levels above 0.002%. It is this PCP ratio “dip”with normals which allows valid comparisons against similar measurementsof APC resistance and PCP defects on plasmas with defects such as thosedue to oral anticoagulants as shown in FIG. 1.

Factor V(Leiden) Confirmation Assay

In order to differentiate between normal and factor V(Leiden) sampleswhich gave “borderline” results in the blood clotting assay according tothe present invention, samples were re-assayed after first being diluted1:1 in distilled water. The results are shown in FIG. 5. The threefactor V(Leiden) samples tested produced lower ratios than those of thenormal samples re-assayed after dilution with water. This finding allowsthe testing of samples without the need to add factor V deficient plasmato the samples to be assayed.

GAG test

This basic test is carried out in two parts: Firstly, a two stageprocedure involving a preincubation, like an APTT and second, a one steptest without preincubations, just like a PT test.

In the 2 stage procedure, test plasma (eg 0.1 ml) is preincubated withan equal volume of factor Xa (optimally 0.002 u/ml or higher if GAGs areadded) in 0.02 M HEPES buffer at pH 7.2 for 5 minutes at 37 degC. Then afurther equal volume(0.1 ml) of 0.02 M calcium chloride containing 0.1%soybean lecithin is added and the time to clot is determined. The targetrange is 80-120 sec for normals.

The above result is compared with that obtained when the same testplasma (0.1 ml) is clotted with 0.2 ml of a 1:1 mixture of the 2reagents (ie. the FXa reagent and the calcium chloride/phospholipidreagent). The target range is 30-35 sec.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

-   BERITNA RM, KEULEMANS BPC, KOSTER T, et al.- Mutation in blood    coagulation factor V associated with resistance to activated    protein C. Nature 369; 64-67, 1994.-   DAHLBACK B - Inherited Thrombophilia: Resistance to activated    protein C as a pathogenic factor of venous thromboembolism. Blood    85; 607-614, 1995-   MITCHELL CA, ROWELL JA, HAU L, et al.- Fatal thrombotic disorder    associated with an acquired inhibitor of protein C. N. England J.    Med. 317; 1638-16, 1987-   AMER L, KISIEL W, SEARLES RP, WILLIAMS JRC - Impairment of the    protein C anticoagulant pathway in a patient with systemic lupus    erythematosus, anticardiolipin antibodies and thrombosis. Thromb.    Res. 57, 247-1990

1. A method of determining the coagulation potential of a plasma sample,the method comprising the steps of: (a) pre-incubating the plasma samplewith a reagent such that (i) endogenous protein C in the plasma is atleast partially converted into activated protein C by the reagent, and(ii) adding factor Xa which is progressively inactivated by antithrombinIII/heparin cofactor 2 during the preincubation; (b) adding an exogenousreagent which activates factor X to Xa or prothrombin to thrombin in afactor V-dependent manner to the preincubated plasma sample of step (a);(c) monitoring a reaction indicative of the rate of coagulation of theplasma sample; (d) comparing the rate of coagulation monitored in step(c) with the equivalent rate determined for an individual withoutimpaired coagulation control, or comparing the rate of coagulationdetected in step (c) with the equivalent rate determined for the plasmasample in the absence of protein C activator; and (e) determining thecoagulation potential of the plasma sample from one or other of thecomparisons of step (d).
 2. The method according to claim 1 wherein thereagent of step (a) further contains low concentrations ofglycosaminoglycans.
 3. The method according to claim 2 wherein theglycosaminoglycans are selected from the group consisting of regular orlow weight heparins, and dermatan or dextran sulphates.
 4. The methodaccording to claim 1 whereby the exogenous agent that transforms proteinC into activated protein C is substantially diluted whole snake venom.5. The method according to claim 4 whereby the snake venom isAgkistrodon species including Agkistrodon Contortrix, or a relatedspecies including A. Piscovorus, A. Bilineatus, A. C Laticinctus, and A.C. Moccason.
 6. The method according to claim 5 wherein the venom is A.Contortrix whole venom and diluted at a concentration of about 0.002%.7. The method according to claim 5 wherein the snake venom is diluted A.Contortrix whole venom.
 8. The method according to claim 1 wherein thepreincubation in step (a) is carried out at neutral or slightly basicconditions.
 9. The method according to claim 8 wherein the preincubationstep is carried out at pH 7.5.
 10. The method according to claim 1whereby the preincubation is carried out for sufficient time foractivation of protein C in the plasma.
 11. The method according to claim10 wherein the preincubation time is about 5 minutes.
 12. The methodaccording to claim 1 whereby the factor Xa is of human or animal origin.13. The method according to claim 1 whereby the exogenous reagent whichactivates factor X to Xa is derived from the venom of Russells Viper(Vipera Russelli) or other immunologically cross-reactive snake species.14. The method according to claim 1 wherein the exogenous reagent whichactivates prothrombin to thrombin in a factor V-dependent manner isderived from Australian Notechis, or Pseudonaja or Oxyuranus snakevenoms.
 15. The method according to claim 14 wherein the snake venom isobtained from the species selected from the group consisting ofPseudonaja Textilis, Notechis Scutatus, and Oxyuranus Scutellatus. 16.The method according to claim 1 whereby reagents in step (b) arecombined with other components into a single mixture by the use ofsurfactants.
 17. The method according to claim 16 wherein thesurfactants are non-ionic detergents.
 18. The method according to claim16 or 17 wherein the single mixture further contains supplementalcomponents selected from the group consisting of buffers, preservatives,hexadimethrine bromide (polybrene) or other agents to reverse the effectof any heparin that may be present in the test samples or which may beadded in the preincubation reagent (i), and phospholipid at high ionicstrength to overcome non-specific inhibitors such as lupusanticoagulants that may be present in the plasma sample.
 19. The methodaccording to claim 1 whereby the monitoring a reaction indicative of therate of coagulation of the plasma sample is a coagulation time assay ora chromometric or uorometric assay using a detectable substrate.
 20. Themethod according to claim 1 wherein phospholipid and calcium ions areadded; to the pre-incubated plasma sample.